Stability enhancement of cytochrome c through heme deprotonation and mutations.
The chemical denaturation of Pseudomonas aeruginosa cytochrome c(551) variants was examined at pH 5.0 and 3.6. All variants were stabilized at both pHs compared with the wild-type. Remarkably, the variants carrying the F34Y and/or E43Y mutations were more stabilized than those having the F7A/V13M or V78I ones at pH 5.0 compared with at pH 3.6 by ~3.0-4.6 kJ/mol. Structural analyses predicted that the side chains of introduced Tyr-34 and Tyr-43 become hydrogen donors for the hydrogen bond formation with heme 17-propionate at pH 5.0, but less efficiently at pH 3.6, because the propionate is deprotonated at the higher pH. Our results provide an insight into a stabilization strategy for heme proteins involving variation of the heme electronic state and introduction of appropriate mutations.
Sonoyama
Takafumi
T
Graduate School of Biosphere Science, Hiroshima University, CREST of Japan Science and Technology Corporation, 1-4-4 Kagamiyama, Higashi-Hiroshima, Hiroshima 739-8528, Japan.
Hasegawa
Jun
J
Uchiyama
Susumu
S
Nakamura
Shota
S
Kobayashi
Yuji
Y
Sambongi
Yoshihiro
Y
D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics*; D045304:Cytochromes c / Q000737:chemistry* / Q000235:genetics*; D019791:Guanidine / Q000009:adverse effects; D006418:Heme / Q000031:analogs & derivatives / Q000737:chemistry*; D006860:Hydrogen Bonding; D006863:Hydrogen-Ion Concentration; D009154:Mutation; D011489:Protein Denaturation; D055550:Protein Stability; D011522:Protons; D011550:Pseudomonas aeruginosa / Q000737:chemistry / Q000235:genetics
D001426:Bacterial Proteins; D011522:Protons; C081433:heme propionate-7; D006418:Heme; D045304:Cytochromes c; D019791:Guanidine